# US 2013/0156743 A1 · Vitro hepatic differentiation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0156743 A1 - Document ID: US20130156743A1 - Status: Published application - Kind: A1 - Application number: 13/818,515 - Filed: 2011-08-25 - Published: 2013-06-20 - Earliest claimed: 2010-08-25 - Assignees: Cambridge Enterprise Limited - Inventors: Ludovic Vallier; Sheikh Tamir Rashid; Nicholas Hannan; Hsin-Hua Cho - Web page: https://patentgrep.com/patent/US20130156743A1 ## CPC - C12N 15/85: for animal cells - C12N 5/067: {Hepatocytes} - C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2) - C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor - C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract This invention relates to the induction of hepatic differentiation by culturing induced pluripotent stem (iPS) cells in an endoderm induction medium to produce a population of anterior definitive endoderm (ADE) cells and then culturing the population of ADE cells in a hepatic induction medium to produce a population of hepatic progenitor cells, which may be optionally differentiated into hepatocytes. The endoderm induction medium is a chemically defined medium which has fibroblast growth factor activity, stimulates SMAD2 and SMAD3 mediated signalling pathways and SMAD1, SMAD5 and SMAD9 mediated signalling pathways, and inhibits phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β); and the hepatic induction medium is a chemically defined medium which stimulates SMAD2 and SMAD3 mediated signalling pathways. These methods may be useful, for example, in producing hepatocytes and hepatic progenitor cells for cell-based therapies or disease modelling. ## Claims ### Claim 1 A method for inducing hepatic differentiation comprising; (i) providing a population of induced pluripotent stem (iPS) cells, (ii) culturing the population in a endoderm induction medium to produce a population of anterior definitive endoderm (ADE) cells, wherein the endoderm induction medium is a chemically defined medium which has fibroblast growth factor activity and which stimulates SMAD2 and SMAD3 mediated signalling pathways and SMAD1, SMAD5 and SMAD9 mediated signalling pathways and inhibits phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β); and, (iii) culturing the population of ADE cells in a hepatic induction medium to produce a population of hepatic progenitor cells, wherein the hepatic induction medium is a chemically defined medium which stimulates SMAD2 and SMAD3 mediated signalling pathways. ### Claim 2 (depends on 1) A method according to claim 1 wherein the endoderm induction medium comprises fibroblast growth factor ### Claim 3 (depends on 1, 2) A method according to claim 1 or claim 2 wherein the endoderm induction medium comprises a first TGFβ ligand which stimulates SMAD2 and SMAD3 mediated signalling pathways. ### Claim 4 (depends on 3) A method according to claim 3 wherein the first TGFβ ligand is activin. ### Claim 5 A method according to any one of the preceding claims wherein the endoderm induction medium comprises a second TGFβ ligand which stimulates SMAD1, SMAD5 and SMAD9 mediated signalling pathways. ### Claim 6 (depends on 5) A method according to claim 5 wherein the second TGFβ ligand is bone morphogenic protein. ### Claim 7 A method according to any one of the preceding claims wherein the endoderm induction medium comprises a PI3K inhibitor and a GSK3β inhibitor. ### Claim 8 (depends on 7) A method according to claim 7 wherein the PI3K inhibitor is LY294002. ### Claim 9 (depends on 7, 8) A method according to claim 7 or claim 8 wherein the GSK3β inhibitor is CHIR99021. ### Claim 10 A method according to any one of the preceding claims wherein the hepatic induction medium comprises a first TGFβ ligand which stimulates SMAD2 and SMAD3 mediated signalling pathways. ### Claim 11 (depends on 10) A method according to claim 10 wherein the first TGFβ ligand is activin. ### Claim 12 A method according to any one of the preceding claims wherein step (ii) comprises: (a) culturing the population of iPSCs in said endoderm induction medium, (b) further culturing the population in the endoderm induction medium lacking GSK3β inhibitor; and, (c) further culturing the population in ADE induction medium which stimulates SMAD2 and SMAD3 signalling pathways and which has fibroblast growth factor activity to produce the population of anterior definitive endoderm (ADE) cells. ### Claim 13 (depends on 12) A method according to claim 12 wherein the ADE induction medium comprises fibroblast growth factor ### Claim 14 (depends on 12, 13) A method according to claim 12 or claim 13 wherein the AE induction medium comprises a first TGFβ ligand which stimulates SMAD2 and SMAD3 mediated signalling pathways. ### Claim 15 (depends on 14) A method according to claim 14 wherein the first TGFβ ligand is activin. ### Claim 16 (depends on 12, 13, 14, 15) A method according to any one of claims 12 to 15 wherein the population is cultured for 24 hours in each of steps a) to c). ### Claim 17 A method according to any one of the preceding claims further comprising; (iv) culturing the population of hepatic progenitor cells in a hepatic maturation medium to produce a population of hepatocytes. ### Claim 18 (depends on 17) A method according to claim 17 wherein the population of hepatic progenitor cells is cultured for 10 to 20 days to produce the population of hepatocytes. ### Claim 19 (depends on 17, 18) A method according to claim 17 or claim 18 comprising determining the production of albumin and/or α1-antitrypsin by the population of hepatocytes. ### Claim 20 (depends on 17, 18, 19) A method according to any of claims 17 to 19 wherein at least 80% of said population of hepatocytes express albumin and/or α1-antitrypsin. ### Claim 21 (depends on 17, 18, 19, 20) A method according to any of claims 17 to 20 comprising expanding the population of hepatic progenitor cells or hepatocytes. ### Claim 22 (depends on 17, 18, 19, 20, 21) A method according to any of claims 17 to 21 comprising culturing or maintaining the population of hepatic progenitor cells or hepatocytes. ### Claim 23 (depends on 17, 18, 19, 20, 21, 22) A method according to any of claims 17 to 22 comprising storing the population of hepatic progenitor cells or hepatocytes. ### Claim 24 A method according to any one of the preceding claims comprising admixing the population of hepatic progenitor cells or hepatocytes with a pharmaceutically acceptable excipient. ### Claim 25 A method according to any one of the preceding claims wherein the iPS cells are human iPS cells. ### Claim 26 A method according to any one of the preceding claims wherein said iPS cells have been passaged 30 times or less. ### Claim 27 A method according to any one of the preceding claims wherein the iPS cells are derived from fibroblasts obtained from an individual. ### Claim 28 A method according to any one of the preceding claims wherein the iPS cells are derived from cells obtained from an individual with damaged or dysfunctional liver tissue and the hepatocytes in the population display a normal phenotype. ### Claim 29 (depends on 28) A method according to claim 28 wherein a liver disease associated genetic mutation or defect in the iPS cells is corrected, such that said hepatocytes display a normal phenotype. ### Claim 30 (depends on 29) A method according to claim 29 wherein the liver disease is an inherited metabolic disorder. ### Claim 31 (depends on 30) A method according to claim 30 wherein the IMD is selected from the group consisting of Alpha 1 Antitrypsin deficiency, a Glycogen Storage Disease, Familial Hypercholesterolemia, Hereditary Tyrosinaemia, Crigler Najjar syndrome, ornithtine transcarbamylase deficiency, factor IX deficiency, haemochromatosis, Wilson's disease, Dubin-Johnson syndrome, familial amyloidosis, and Refsum's disease. ### Claim 32 (depends on 29) A method according to claim 29 wherein the genetic mutation or defect is a Glu342Lys mutation in A1AT and the liver disease is alpha 1 antitrypsin deficiency. ### Claim 33 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) A method according to any one of claims 1 to 27 wherein the iPS cells are derived from cells obtained from an individual with a liver disease and the hepatocytes in the population display a disease phenotype. ### Claim 34 (depends on 33) A method according to claim 33 wherein the liver disease is an inherited metabolic disorder. ### Claim 35 (depends on 34) A method according to claim 34 wherein the IMD is selected from the group consisting of Alpha 1 Antitrypsin deficiency, a Glycogen Storage Disease, Familial Hypercholesterolemia, Hereditary Tyrosinaemia, Crigler Najjar syndrome, ornithtine transcarbamylase deficiency, factor IX deficiency, haemochromatosis, Wilson's disease, Dubin-Johnson syndrome, familial amyloidosis, and Refsum's disease. ### Claim 36 (depends on 33, 34, 35) A method according to any one of claims 33 to 35 comprising detecting or measuring one or more disease pathologies in the population of hepatocytes. ### Claim 37 (depends on 36) A method according to claim 36 wherein the disease pathologies are detected by measuring or determining one or more of protein aggregation or polymerisation; protein entrapment in the ER; cholesterol uptake; lipid and/or glycogen accumulation; and lactic acid production in said population of hepatocytes. ### Claim 38 (depends on 33, 34, 35, 36, 37) A method according to any one of claims 33 to 37 comprising correcting a genetic mutation or defect in the hepatocytes in the population, such that said hepatocytes display a normal phenotype. ### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38) A population of isolated hepatic progenitors or hepatocytes produced by a method according to any one of claim 1 to 35 or 38, wherein said progenitors or hepatocytes display a normal phenotype. ### Claim 40 (depends on 39) A population according to claim 39 for use in a method of treatment of the human or animal body. ### Claim 41 (depends on 40) A population according to claim 40 for use in a method of treatment of a patient with damaged or dysfunctional hepatic tissue. ### Claim 42 (depends on 36, 37) A population of hepatocytes produced by a method according to any one of claims 36 to 37, wherein said hepatocytes display a disease phenotype. ### Claim 43 (depends on 39) A method of treating a patient with a damaged or dysfunctional hepatic tissue comprising; administering a population of hepatocytes according to claim 39 to an individual in need thereof. ### Claim 44 (depends on 39) Use of a population according to claim 39 in the manufacture of a medicament for use in the treatment of a patient with a damaged or dysfunctional hepatic tissue. ### Claim 45 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) A method of screening a compound comprising; contacting isolated hepatocytes produced by a method according to any one of claims 1 to 38 with a test compound, and; determining the effect of the test compound on said hepatocytes and/or the effect of said hepatocytes on the test compound. ### Claim 46 (depends on 45) A method according to claim 45 comprising determining the growth or viability of the hepatocytes or the ability of the hepatocytes to perform one or more hepatocyte functions in the presence relative to the absence of the test compound, wherein a decrease in growth, viability or ability to perform one or more hepatocyte functions is indicative that the compound has a hepatotoxic effect. ### Claim 47 (depends on 46) A method according to claim 46 wherein the metabolism of the test compound by the hepatocytes is determined. ### Claim 48 (depends on 47) A method according to claim 47 comprising determining or measuring decreases in the amount or concentration of test compound and/or increases in the amount or concentration of an metabolite of said test compound. ### Claim 49 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) A method of screening for a compound useful in the treatment of an liver disease comprising; contacting a population of hepatocytes produced by a method according to any one of claims 1 to 38 with a test compound, and; determining the effect of the test compound on said hepatocytes. ### Claim 50 (depends on 49) A method according to claim 49 wherein the hepatocytes display a disease phenotype and the effect of the test compound on one or more disease pathologies in the hepatocytes is determined. ### Claim 51 (depends on 50) A method according to claim 50 wherein the effect of the test compound on one or more of protein aggregation or polymerisation; protein entrapment in the ER; cholesterol uptake; lipid and/or glycogen accumulation; and lactic acid production is determined. ### Claim 52 (depends on 50, 51) A method according to any one of claims 50 to 51 wherein a decrease or amelioration of one or more disease pathologies in the hepatocytes in the presence, relative to the absence of test compound is indicative that the test compound may be useful in the treatment of a liver disease. ### Claim 53 (depends on 49) A method according to claim 49 wherein the hepatocytes display a normal phenotype and the effect of the test compound on the ability of the hepatocytes to perform one or more hepatocyte functions is determined. ### Claim 54 (depends on 53) A method according to claim 53 wherein the one or more hepatocyte functions are selected from detoxification, glycogen storage, secretion of AAT or albumin, bile production, thrombopoietin production, angiotensinogen production, conversion of ammonia to urea, cholesterol synthesis, glycogenolysis, glycogenesis and lipogenesis. ### Claim 55 (depends on 53, 54) A method according to any one of claims 53 to 54 wherein an increase in the ability of the hepatocytes to perform one or more hepatocyte functions in the presence, relative to the absence of test compound is indicative that the test compound may be useful in the treatment of a liver disease.